β-galactosidases hydrolyze non-reducing terminal β-D-galactose in β-D-galactopyranosides such as lactose to make galactose and glucose or catalyze the transition of non-reducing galactose to other compounds. Generally, such enzymes have two functions (hydrolytic activity and transglycosylation activity), but the ratio of the two reactions varies depending on the kind of β-galactosidase. The hydrolytic activity of β-galactosidases hydrolyzes lactose in milk and dairy products to prevent lactose intolerance and is used to increase the sweetness of milk or produce sweet syrup as a milk byproduct. The transglycosylation activity of β-galactosidases is used for the production of galactooligosaccharides which stimulate the growth of lactic acid bacteria that are human intestinal beneficial microorganisms. Thus, β-galactosidases are industrially very useful enzymes.
Beta-galactosidases are widely found in mammalian organs, plant seeds, bacteria, fungi, and yeasts. In the food industry, beta-galactosidases from yeasts such as Kluyveromyces lactis and Kluyveromyces fragilis, fungi such as Aspergillus niger and Aspergillus oryzae, and bacteria such as Bacillus circulans, have been used. Among them, beta-galactosidase from Bacillus circulans ATCC 31382 is commercially available under the trade name of Biolacta (Daiwa Kasei, U.S. Pat. No. 4,237,230 (1980)).
Beta-galactosidase bonds with lactose and a reaction begins while the carboxyl group of a glutamate residue in the reaction center acts as an acid/base catalyst (Juers et al., Biochemistry, 40, 14781-14794, 2001). This nucleophile glutamate residue attacks carbon 1 of galactose bound to glucose to detach the glucose and form a temporary covalent bond with the galactose. A hydroxyl group attached to carbon 4 of the glucose that is detached is stabilized with the aid of other glutamates functioning as an acid catalyst and then is detached from the enzyme while it is converted to free glucose by receiving a proton. The galactose forming a temporary covalent bond with the glutamate residue of the enzyme is detached from the enzyme by reaction with water (hydrolysis), or bonds to lactose or other compounds newly introduced into the reaction center of the enzyme (transgalactosylation). A substance having one or more galactose units (galactosides) produced by this transgalactosylation is known as galactooligosaccharide (GOS).
GOS acts as prebiotics that reaches the large intestines without being digested and absorbed, thereby promoting the growth and activity of intestinal useful microorganisms such as Bifidobacteria or Lactobacilli. GOS is known to have health promotion effects, including cancer prevention, mineral absorption, lipid metabolism, anti-inflammation, atopy relief and the like (Macfarlane et al., J. Appl. Mcriobiol., 104, 305-344, 2008). Furthermore, it has been reported that, when a person suffering from irritable bowel syndrome takes GOS, the level of the beneficial intestinal bacteria Bifidobacteria is increased and symptoms of the syndrome are also alleviated (Silk et al., Aliment. Pharmacol. Ther., 29, 508-518, 2009).
Currently, beta-galactosidases from Bacillus or Aspergillus are frequently used for the production of GOS. Particularly, beta-galactosidases from Bacillus circulans are most frequently used for commercial purposes due to their optimum activation temperature (50 to 60° C.), which is relatively high, and their high transglycosylation activity. Various beta-galactosidases, such as 240 kDa beta-galactosidase, 160 kDa beta-galactosidase (Mozafar et al., Agric. Biol. Chem., 48, 3053-3061, 1984); 212 kDa, 145 kDa, 86 kDa (Vetere and Paoletti, Biochim. Biophys. Acta, 1380, 223-231, 1998); 195 kDa, 160 kDa, 135 kDa, 86 kDa (Song et al., Biosci. Biotechnol. Biochem., 75, 268-278, 2011) and the like, derived from Bacillus circulans, were reported. In addition, it has been reported that new beta-galactosidase BgaII having a size of 145 kDa was found in Bacillus circulans (Korean Patent Registration No. 1,121,161).
Accordingly, the present inventors have made extensive efforts to develop a beta-galactosidase having an enhanced ability to produce galactooligosaccharide from lactose, and as a result, have found that, when a mutant was constructed by deleting the C-terminus of beta-galactosidase BgaII, it has an enhanced ability to produce galactooligosaccharide and has dramatically increased thermal stability, thereby completing the present invention.